Novel ovarian endometriosis model causes infertility via iron-mediated oxidative stress in mice

Abstract

Ovarian endometriosis (OE) provides women of reproductive age with not only severe menstrual pain but also infertility and an increased risk for ovarian carcinogenesis. Whereas peritoneal endometriosis models have been developed with syngeneic implantation of minced uterine tissue and oncogenic K-ras allele with conditional Pten deletion within ovarian surface epithelium generated preneoplastic endometrial glandular morphology, followed by endometrioid adenocarcinoma, there has been no mouse model of OE similar to human counterparts, applicable to preclinical studies. Here we for the first time established a murine OE model that reveals infertility, and evaluated the involvement of iron catalyzed oxidative stress in the pathogenesis. Minced uterine tissue from female mice was implanted on ovarian surface of syngeneic mice after bursectomy to induce OE. Ectopic growth of endometrium was observed in association with ovary 4 weeks after implantation in 85.7% (12/14) of the operated mice with our protocol. Endometriotic lesions involved intestine, pancreas and peritoneal wall. Fibrosis around the ovary was prominent and increased time-dependently in the OE group. Iron accumulation was significantly increased in the OE group, leading to oxidative stress in each stage of the follicles as evaluated by 4-hydroxy-2-nonenal-modified proteins and 8-hydroxy-2'-deoxyguanosine. Expression of follicle stimulating hormone receptor in the follicles revealed a significant decrease during pre-antral, antral and pre-ovulatory phases in the OE group. Finally, the number of pups was significantly reduced in the OE group in comparison to the controls. This model affords an opportunity to evaluate agents or procedures to counteract ovarian endometriosis in the preclinical settings.

Keywords: Follicle stimulating hormone receptor; Infertility; Iron; Mouse model; Ovarian endometriosis; Oxidative stress.

Graphical abstract

Fig. 1

Protocol to generate the ovarian endometriosis model in mice. (A) Summary of experimental design. (B) Experimental procedures. C57BL/6 N female mice at 9 weeks of age were used. Donor mice were euthanized and the dissected uterus was longitudinally cut and minced at ~0.5 mm in diameter with scissors. Minced uterus was incubated with a collagenase solution (1 mg/ml) for 30 min at 37 °C, which was centrifugated (8000×g, 5 min) to remove supernatant, and resuspended in 100 μl of phosphate-buffered saline (PBS) to be rinsed by an additional centrifugation (8000×g, 5 min). Then, the tissue pellet was used for transplantation. Recipient mice at 9 weeks of age under isoflurane anesthesia received 5 mm bilateral incisions on their lower back and the ovaries were exposed, to which 100 μl of PBS was injected to intra bursal cavity to identify bursal membrane. Then, we cut it off to expose the ovarian surface. A half of uterine pellet was attached on each exposed ovarian surface. The ovaries were pushed back into the peritoneal cavity, followed by suture of the dorsal scissions. Refer to text for details.

Fig. 2

Analysis of the novel ovarian endometriosis model. Ectopic endometrial cystic growth was visible in association with ovary 4 weeks after the transplantation in the OE (ovarian endometriosis) group. The mice in each group were euthanized 1, 2 and 4 weeks after the procedure and the lesions were evaluated. (A) Single or multiple cystic lesions were observed in association with the ovaries of the OE group whereas those in the other groups were not altered. Adhesion was evident between the endometriotic cysts and other adjoining organs, such as intestine, pancreas and abdominal wall. (B, C) Histology confirmed endometriosis. The ovarian cystic lesions of the OE group consisted of single-layered epithelium and underlying stoma with hemosiderin accumulation and CD10 immunopositivity in the endometriotic stroma (arrows; bar = 100 μm). Refer to text for details.

Fig. 3

Fibrosis and iron accumulation in ovarian endometriosis. (A) Fibrotic area was significantly increased in the OE group in comparison to C and S group. (B) Quantitative analysis of A. (C) Fibrosis was time-dependently increased in the OE group. (D) Quantitative analysis of C. (E) Berlin Blue staining revealed hemosiderin deposition only in the OE group at 4 weeks. (F) Quantitative analysis of E (means ± SEM; **, P < 0.01; ***, P < 0.001; n. s., not significant; bar = 100 μm). Refer to text for details. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

High lipid peroxidation levels as evaluated by 4-hydroxy-2-nonenal-modified proteins (4-HNE) in the primordial, preantral and antral stages of ovarian follicles of ovarian endometriosis. (A) Representative 4-HNE immunohistochemical results of ovarian follicles in each group. Premordial preantral and antral follicles revealed higher levels of 4-HNE in the OE group in comparison to C and S groups. (B- F) Quantitative analysis of each follicular stage of the ovary (means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; n. s., not significant; bar = 20 μm). Refer to text for details.

Fig. 5

High levels of nuclear 8-hydroxy-2′-deoxyguanosine (8-OHdG) in the granulosa cells during the whole follicular stages of ovarian endometriosis. (A) Representative 8-OHdG immunohistochemical results of ovarian follicles in each group. Follicles of all the stages revealed significantly higher levels of nuclear 8-OHdG in the OE group in comparison to C and S groups. (B- F) Quantitative analysis of each follicular stage of the ovary (means ± SEM; *, P < 0.05; **, P < 0.01; n. s., not significant; bar = 20 μm). Refer to text for details.

Fig. 6

Follicle stimulating hormone receptor (FSHR) expression was significantly decreased in ovarian endometriosis. (A) Representative FSHR immunohistochemical results of ovarian follicles at the preantral, antral and preovulatory stages in each group. Follicles of all the stages examined revealed significantly lower levels of FSHR in the OE group in comparison to C and S groups. (B- F) Quantitative analysis of each follicular stage of the ovary (means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; n. s., not significant; bar = 20 μm). Refer to text for details.

Fig. 7

Subfertility in the ovarian endometriosis model. (A) Representative results of mating experiments in each group. The number of pups in the OE group (n = 4) was significantly lower than those in C (n = 3) and S (n = 4) groups. (B) Quantitative analysis of A (means ± SEM; *, P < 0.05). Refer to text for details.